Gene therapy model for stromal precursor cells of hematopoietic microenvironment.

Marker bacterial Neor gene was transduced by retroviral gene transfer into stromal precursor cells making up the hematopoietic microenvironment in murine long-term bone marrow cultures (LTBMC). Cultures were infected six times during the first 3 weeks of cultivation. At 4 weeks, the adherent cell layers (ACLs) were implanted under the renal capsule of syngeneic unirradiated and irradiated mice. Cells from newly formed ectopic foci were explanted into secondary LTBMC. ACLs containing the marker gene were detected by polymerase chain reaction. About 74% of stromal cells in ACLs contained Neor gene. The possibility of stable gene transduction into stromal precursor cells competent to transfer the hematopoietic microenvironment was established.

Individual clones of hemopoietic cells in murine long-term bone marrow culture.

Forty-seven individual hemopoietic cell clones bearing unique radiation markers were studied in long-term bone marrow cultures. Throughout cultivation clones appeared at different times, from 1 to 12 weeks after explantation, survived during 1-10 more weeks, and were characterized by marked variability in size. Usually, the number of metaphases peculiar to an individual clone rapidly increased, achieved maximum, and then underwent a decline. Cells of reliably disappearing clones were never seen again. The experimental results provide further evidence for the model of hemopoiesis by clonal succession.

Effect of hemic cells on hybrid resistance.

Injection of various parental strain hemic cells reduces the ability of the F1 hybrid to resist growth of grafted hemopoietic tissue from the same parent strain. Such an effect can be exerted by cells from lymph nodes, spleen, bone marrow, or thymus, the peritoneal macrophages, and the peripheral blood leukocytes. Embryonic and adult liver cells or blood erythrocytes do not inhibit hybrid resistance. It is assumed that hemic cells inhibit hybrid resistance through an effect on the hemopoietic microenvironment. The mechanism of this effect cannot be considered the result of a graft-versus-host reaction since lymphoid cells which are "immune" or "tolerant" to the hybrid antigens possess inhibitory activity similar to that of intact lymphoid cells. The hybrid resistance inhibitory cells maintain the inability of "parent-in-F1 hybrid" type chimeras to express hybrid resistance for at least 2 months.

Role of bone marrow stroma in hemopoietic stem cell regulation.

Bone marrow plugs from C57Bl mice were irradiated with 1500 rad, implanted under the kidney capsule of (CBA x C57Bl)F1 hybrid mice and reimplanted one month later in identical F1 hybrid mice. Foci of ectopic hemopoiesis formed in which all hemopoietic cells were of recipient origin, while the stroma was from the donor bone marrow. The hybrid resistance in such foci was of donor origin. Implantation of 8--10 femoral plugs under the capsule of each kidney produced large foci of ectopic hemopoiesis containing thousands of hemopoietic stem cells (CFUS). The sum total CFUS content in such mice was two- to four-fold than in normal mice in steady-state hemopoiesis. It is concluded that the regulation of stem cell proliferation and total content in the body is governed by the stroma of hemopoietic organs.

Hemopoietic stromal precursors in long-term culture of bone marrow: II. Significance of initial packing for creating a hemopoietic microenvironment and maintaining stromal precursors in the culture.

Conversion of bone marrow cells to a single cell suspension prevents them from creating a hemopoiesis maintaining adherent cell layer (ACL) in culture. The ACL of such cultures is devoid of hemopoietic stromal precursors capable of transferring the hemopoietic microenvironment on implantation under the renal capsule of syngeneic mice. The regeneration of ACL after injury is possible in 1-wk-old, but not older, Dexter-type bone marrow cultures. The results prove the significance of the initial packing of the bone marrow cells in the differentiation of the hemopoietic stromal precursors. These data contradict previous reports which concluded that the structural orderliness of stromal tissue need not be preserved for the hemopoietic microenvironment to be transferred.

Self-maintenance ability of circulating hemopoietic stem cells.

In adult mice, circulating hemopoietic stem cells (CFUs) and their progeny that repopulate an ectopic hematopoietic site possess a self-maintenance potential that is less than that of resident stem cells. This is manifested by a decrease in the content of daughter CFUs in 11-day spleen colonies produced by circulating CFUs, compared to those produced by resident CFUs. Study of the CFUs content in 139 individual spleen colonies showed that circulating CFUs produce the entire spectrum of colonies, both those with a low and those with a high CFUs content. However, the proportion of colonies that contained 10 or fewer CFUs was much higher among the colonies produced by circulating CFUs (60-70%) than by the resident CFUs (less than 10%). The lower self-maintenance of circulating CFUs, compared to that of resident CFUs, is also confirmed by direct comparison of the overall regeneration of hemopoietic populations derived from blood and bone marrow by means of chromosome markers. The embryonal circulating and resident CFUs (from the liver) do not differ in self-maintenance. In adult mice, intravenously infused circulating CFUs leave the circulation no more slowly than do the resident CFUs.

Hemin stimulation of hemopoiesis in murine long-term bone marrow culture.

The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.

Origin of hemopoietic stromal progenitor cells in chimeras.

Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.

Hemopoietic stromal precursors in long-term culture of bone marrow: I. Precursor characteristics, kinetics in culture, and dependence on quality of donor hemopoietic cells in chimeras.

The adherent cell layer of long-term Dexter type bone marrow culture contains hemopoietic stromal precursors capable of transferring the hemopoietic microenvironment on transplantation in vivo. The foci of ectopic hemopoiesis that are created correspond to normal bone marrow in the character of the maintained hemopoiesis. Radiosensitivity of the cultured stromal precursors does not differ from preculture sensitivity. Stromal precursors lose osteogenic potential after 5-6 wk in culture, but are capable of creating stable foci of ectopic hemopoiesis composed of up to 10(8) nucleated hemopoietic cells. These results prove that bone is not an essential component of the hemopoietic environment in ectopic foci. In chimeras exposed to equal doses of radiation but reconstituted by freshly isolated or repeatedly passaged bone marrow, the content of stromal precursors is lower when hemopoietic cells of diminished self-maintenance are used. These data suggest the existence of a hemopoietic stromal precursor hierarchy with at least two levels of precursor differentiation.

Long-term bone marrow stromal and hemopoietic toxicity to AZT: protective role of heme and IL-1.

We studied the immediate and long-term effects of azidothymidine (AZT) and heme on murine hemopoietic and stromal progenitor cells in vivo and in vitro. Treatment of mice for 37 days with AZT produced anemia and leukopenia, whereas combined treatment with heme abrogated some of the toxic effects which were apparent even 2 weeks after cessation of treatment. Quantitation of spleen (CFU-S), erythroid (BFU-E) and myeloid (CFU-GM) colony formation from AZT-exposed animals revealed reductions in these progenitors, and this was partially reversed after heme treatment, especially when mice were allowed a 2-week recovery period. Long-term bone marrow cultures (LTBMC) of cells from treated groups revealed difficulty in establishing an adherent cell layer (ACL) by the first week in culture. Total cellularity, CFU-S, BFU-E and CFU-GM clonogenic potential of cultures remained depressed throughout 10 weeks of culture, whereas heme treatment overcame these depressions when AZT-exposed mice were allowed to recover for 14 days prior to culture of their cells in LTBMC. Interleukin-1 (IL-1) treatment to the same recovery group of AZT-exposed mice also resulted in an improvement of CFU-GM growth in LTBMC that was not seen in the nonrecovered group. Transplantation of cells from treated mice under the renal capsule of recipient mice revealed that AZT depressed the regeneration of osteogenic and hemopoietic cell growth within ectopic foci. These effects were reversed with heme treatment in vivo. In other experiments, heme was found to inhibit human immunodeficiency virus (HIV-1) reverse transcriptase and to potentiate the activity of AZT triphosphate against HIV-1 reverse transcriptase. In summary, these results demonstrate that AZT inhibits the growth and development of a variety of hemopoietic, stromal and adherent cells in vivo and in vitro. Treatment of animals with heme produced recovery to near normal levels and suggests possible therapeutic potential.

Lifelong hematopoiesis in both reconstituted and sublethally irradiated mice is provided by multiple sequentially recruited stem cells.

OBJECTIVE: To evaluate the dynamics of stem cell production to hematopoiesis, the number of active stem cell clones and the lifespan of individual clones were studied. MATERIALS AND METHODS: The clonal contribution of primitive hematopoietic stem cells (HSC) responsible for long-term hematopoiesis was determined using two approaches. In one model, irradiated female mice were reconstituted with retrovirally marked male hematopoietic cells. In the second model, mice were irradiated sublethally without hematopoietic cell transplantation. In both models, bone marrow cells were serially sampled from the same mouse throughout a 12- to 20-month period and injected into irradiated recipients for analysis of day 10 colony-forming unit-spleen (CFU-S). The donor origin of CFU-S was determined by the presence of retrovirally marked cells or cells with chromosomal aberrations. RESULTS: The results of the two essentially different models show that 1) hematopoiesis is mainly the product of small clones of hematopoietic cells; 2) the lifespan of the majority of clones is only 1 to 2 months; 3) the clones usually function locally; and 4) the vast majority of the clones replace one another sequentially. Primitive HSCs capable of producing long-lived clones (about 10% among all clones), which exist during the entire life of a mouse, were detected by the radiation-marker technique only. CONCLUSION: Multiple short-living clones (at least on the level of CFU-S production) comprise the vast majority of the active stem cells in transplanted recipients or after endogenous recovery from sublethal irradiation.

Cells responsible for restoration of haemopoiesis in long-term murine bone marrow culture.

Long-term haemopoiesis in bone marrow culture is sustained by the progeny of haemopoietic progenitor cells (HPC), which differ from CFUs by very low sensitivity to repeated hydroxyurea (HU) injections. The transit time of a haemopoietic clone from HPC to cells proliferating in culture is 6-7 weeks. The results suggest that the stem cell continuum is an expansion type compartment, members of which gradually lose their proliferative potential during differentiation.

Comparative effect of heme analogues on hematopoiesis in lymphoproliferative disorders.

Anemia is a common characteristic of lymphoproliferative disorders (LPD) and the impairment of blood formation in these disorders is not fully understood. Heme synthesis and the heme degradative enzyme heme oxygenase are critical to hematopoietic differentiation and disturbances may contribute to anemic states. Tin protoporphyrin (SnPP) is a potent inhibitor of heme oxygenase, and has proven to be a useful clinical agent. Bone marrow cells from seven patients with LPD were studied for their in vitro hemopoietic response to growth factors and SnPP. Heme oxygenase mRNA levels were determined by Northern blot analysis of bone marrow samples. Quantitation of hematopoiesis in cultures with erythropoietin or GM-CSF revealed adequate CFU-E, BFU-E and CFU-GM growth by LPD bone marrow. Inclusion of 10 µM SnPP in cultures was found to significantly enhance CFU-E/BFU-E growth by LPD marrows, whereas Zinc protoporphyrin had a marked inhibitory effect. Little or no effect by SnPP was seen on CFU-GM. In contrast, normal bone marrow cultures failed to show an enhanced response to 10 µM SnPP. Analysis of heme oxygenase mRNA levels revealed that LPD marrows had elevated expression of heme oxygenase mRNA as contrasted with normals. Furthermore, measurements revealed that heme oxygenase activity was markedly suppressed by SnPP in the LPD bone marrow cultures. Results lend further support to the importance of heme oxygenase in the differentiation process. Although LPD bone marrow cells may respond to erythropoietin in vitro, in stressed conditions where heme oxygenase is elevated, suppression of heme oxygenase may potentiate the erythropoietic response in this disease.

Transplantation of allogeneic bone marrow from thymus-deprived monkeys and certain characteristics of "graft-versus-host" cells.

Transplantation of allogeneic bone marrow both from normal and thymus-deprived donors (thymectomized lethally irradiated and restored with autologous bone marrow) was followed 6--7 days later by the appearance of large numbers of GVH cells in bone marrow and peripheral blood which were one of the characteristic features of acute GVHR. GVH cells isolated from peripheral blood 7 days after transplantation showed a high labelling index (about 30%) after 40 min incubation with 3H-thymidine. The prolongation of incubation failed to increase the proportion of labelled cells due to the low viability of GVH cells. The results obtained with T and B cell tests suggested that GVH cells had the properties of T lymphocytes: about 60% of the cells could form rosettes with non-sensitized sheep red blood cells; there were virtually neither Ig-bearing cells nor cells with receptors for C3 and Fc portion of Ig.

Immunologic study of T and B lymphocytes in thymectomized monkeys after irradiation and autologous bone marrow transplantation.

Rhesus monkeys were exposed to total-body gamma-irradiation (60Co) with 700R following adult thymectomy, and then protected with autologous bone marrow. Transplantation immunity in TxIBM animals was impaired: skin allografts survived for 19 to 38 days. The percentage of T and B lymphocytes studied by the rosette technique and immunofluorescent staining for Ig did not substantially change when examined in early as well as late periods after TxIBM. Functional capacities of lymphocytes were measured in vitro by stimulation with phytohaemagglutinin and with allogeneic cells in mixed lymphocyte cultures. MLC reactivity in TxIBM monkeys did not recover in the early and later periods following treatment. PHA responsiveness was retained in both these periods. These findings together with the results obtained in the analysis of unstable chromosomal aberrations suggested that PHA-responsive and MLC-reactive cell subpopulations of monkey blood lymphocytes differed in the degree of thymus dependence.